Smoking is the most common preventable cause of morbidity and mortality in the United States. For 90% of adult smokers, this addiction began in adolescence. Effective interventions to block the escalation of this initial smoking in adolescents exist. However, the implementation and effectiveness of these interventions are hindered by the inability of current biomarkers to detect periodic use of cigarettes. This substantial problem has recently made substantially worse by the introduction of e-cigarettes, whose active ingredient, nicotine, is also metabolized to cotinine. As result, clinicians cannot determine which adolescents are smoking nor can scientists reliably determine whether e-cigarette usage is leading to increased rates of adolescent smoking. Recently, using a blood based approach; Behavioral Diagnostic, Inc (BDI) has developed a epigenetic biomarker, referred to as Smoke Signature(tm), that can sensitively and specifically detect cigarette smoking consumption even in the face of e-cigarette use in adults. This patented biomarker assesses the methylation level at a CpG residue in the aryl hydrocarbon receptor referred to as cg05575921 to determine smoking status. The robust sensitivity and specificity of this response of this locus to smoking has been demonstrated in over 40 publications by ourselves and others using tens of thousands of samples. However, since almost all the previous studies have been performed using DNA isolated from blood, and the exact shape of the dose response curve in saliva DNA is not known, we are unable to push forward with our efforts to develop a saliva based kit that could be used 1) scientifically to evaluate the effects of e-cigarettes and 2) clinically to screen adolescents for behavioral intervention and monitor their response to treatment. In Phase I of this Fast Track application, we will take advantage of 1) our already developed quantitative PCR (qPCR) assays, 2) data showing that cg05575921 methylation in blood and saliva is highly correlated, and 3) our already tested saliva kit ingredients to confirm the feasibility of our salia based approach and the equivalence of our saliva DNA kits. In Phase II, we will determine the sensitivity and specificity of our method for detecting smoking using a two qPCR marker set that includes Smoke Signature and a second marker that compensates for the cellular heterogeneity of saliva. Our plan is highly feasible because the expertise of BDI's expertise in epigenetics, ou possession of intellectual property rights and our strong team of commercial collaborators that includes established companies to manufacture and co-market the products (IBI Scientific and IDT). It is innovative because the techniques are state of the art and quantitative epigenetic tests are new to the market. As a direct result of this research we will produce a kit suitable for researchers interested in smoking and the effects of e-cigarettes on the rate of smoking while gaining valuable data that can be used in a future FDA application.